Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative diseases that include such human disorders as sporadic and familial Creutzfeldt-Jakob disease (CJD), kuru, fatal familial insomnia, and Gerstmann-Straussler-Scheinker syndrome. Animal forms of the diseases include scrapie in sheep and bovine spongiform encephalopathy in cattle. These diseases are characterized by the formation and accumulation in the brain of an abnormal proteinase K resistant isoform (PrPres) of a normal protease-sensitive host-encoded prion protein (PrPsen). PrPres is formed from PrPsen by a post-translational process involving conformational changes that convert the PrPsen into a PrPres molecular aggregate having a higher .beta.-sheet content. The formation of these macromolecular aggregates of PrPres is closely associated with TSE-mediated brain pathology in which amyloid deposits of PrPres are formed in the brain, which eventually becomes "spongiform" (filled with holes).
In the past, the TSE diseases were a medical curiosity because the transmissible agent was difficult to inactivate with heat, radiation or chemicals that would be expected to inactivate infectious living organisms such as bacteria and viruses. Instead, this class of diseases appeared to be transmitted by exposure to an unusual agent, for example by ritual cannibalism in the Foret people of New Guinea, or feeding of animal parts to cattle in bovine spongiform encephalopathy (BSE). Iatrogenic CJD has also been caused by administration of human growth hormone derived from cadaveric pituitaries, transplanted dura mater and corneal grafts, as well as exposure of surgeons to affected tissue during neurological procedures. The TSE diseases took on new urgency, however, when it appeared that cross-species infection of humans in Europe may have occurred, perhaps from the ingestion of beef from affected cows. That development has further stimulated an international search for a better understanding of the pathophysiological mechanism of the disease, and possible treatments.
The presence of a native prion protein (PrP) has been shown to be essential to pathogenesis of TSE. The cellular protein PrPsen is a sialoglycoprotein encoded by a gene that in humans is located on chromosome 20. The PrP gene is expressed in neural and non-neural tissues, with the highest concentration of its mRNA being in neurons. The translation product of the PrP gene consists of 253 amino acids in humans, 254 in hamsters and mice, 264 amino acids in cows, and 256 amino acids in sheep (all of these sequences are disclosed in U.S. Pat. No. 5,565,186, which describes methods of making transgenic mice that express species specific PrP. Other sequence information is included in Locht, C. et al., Proc. Natl. Acad. Sci. USA 83:6372-6376, 1986; Kretzschmar, H. A. et al., DNA 5:315-324, 1986; Yoshimoto, J. et al., Virus Genes 6:343-356, 1992; Goldmann, W. et al. Proc. Natl. Acad. Sci. USA 87:2476-2480, 1990). In prion protein related encephalopathies, the cellular PrPsen is converted into the altered PrPres that is distinguishable from PrPsen in that PrPres (1) aggregates; (2) is proteinase K resistant in that only approximately the N-terminal 67 amino acids are removed by proteinase K digestion under conditions in which PrPsen is completely degraded; and (3) has an alteration in protein conformation in which the amount of .alpha.-helical conformation for PrPsen is reduced, and the amount of .beta.-sheet conformation for PrPres is increased.
If PrPsen is not expressed in the brain tissue of animal recipients of scrapie-infected neurografts, no pathology occurs outside the graft, demonstrating that PrPres and PrPsen are both required for the pathology (Brander et al., Nature 379:339-343, 1996). The long latency period between infection and the appearance of disease (months to decades depending on species) has prompted the development of a cell-free in vitro test, in which PrPres induces the conversion of PrPsen to PrPres (Kocisko et al., Nature 370:471-474, 1994). See also Prusiner et al., WO 97/16728 published May 9, 1997. The in vitro interaction between PrPres and PrPsen occurs with species and strain specificities that mimic TSE species barrier effects and strain differences in vivo (Kocisko et al., Proc Natl Acad Sci USA 92, 3923-3927, 1995; Bessen et al., Nature 375, 698-700, 1995; Bossers et al., Proc. Natl. Acad. Sci. USA 94, 4931-4936, 1997; Raymond et al., Nature 388, 285-288, 1997), hence in vitro cell free culture techniques are considered to accurately predict pathological developments in the brains of infected animals. These in vivo and in vitro observations indicate that direct interactions between PrPres and PrPsen form PrPres and promote TSE pathogenesis.
Small synthetic peptides containing certain PrP sequences have previously been shown to spontaneously aggregate to form fibrils with a high degree of .beta.-sheet secondary structure of the type seen in the insoluble deposits in TSE afflicted brains (Gasset et al. Proc. Natl. Acad. Sci. USA 89, 10940-10944, 1992; Come et al., Proc. Natl. Acad. Sci. USA 90, 5959-5963, 1993; Forloni et al., Nature 362, 543-546, 1993; Hope et al., Neurodegeneration 5, 1-11, 1996). Moreover, other synthetic PrP peptides have been shown to interact with PrPsen molecules to form an aggregated complex with increased protease-resistance (Kaneko et al., Proc. Natl. Acad. Sci. USA 92, 11160-11164, 1995; Kaneko et al., J. Mol. Biol. 270, 574-586, 1997). The PrP derived synthetic peptide Ala Gly Ala Ala Ala Ala Gly Ala (SEQ. ID. NO. 1) from positions 113 to 120 of the PrP peptides has been described as the most highly amyloidogenic peptide in the protein (Gasset, M., et al., Proc. Natl. Acad. Sci. USA 89, 10940-10944, 1992), which indicates that it would be expected to promote the formation of PrPres.
Holscher et al. (J. Virol. 72: 1153-1159, 1998) recently showed that a mutant mouse PrP lacking the sequence from 114 to 121 (spanning the highly amyloidogenic region) is not converted into a proteinase K-resistant isoform after expression in scrapie-infected mouse neuroblastoma cells. This finding further supported the idea that this highly hydrophobic sequence promoted PrPres formation. U.S. Pat. No. 5,618,673 disclosed a scrapie specific palindromic oligonucleotide that hybridized to the DNA of scrapie infected tissue for use in diagnostic assays. U.S. Pat. No. 5,679,530 described a prion binding protein and peptide that were said to be useful in the non-histologic diagnosis of prion diseases.
Although these discoveries have supported the grim suggestion that certain PrP sequences can promote the formation of insoluble PrP deposits in the brain and elsewhere in the body, they have done little to provide an approach for inhibiting the formation of such deposits. However, U.S. Pat. No. 5,276,059 disclosed that mammalian diseases associated with amyloid protein formation, and the conversion of PrPsen to PrPres, could be treated by administering Congo Red dye to the mammal. U.S. Pat. No. 5,134,121 disclosed the use of a nerve growth blocking peptide to treat prion associated diseases.
Nonetheless, the need still remains for agents that will specifically inhibit the formation of PrPres, and by extension prevent or slow the deposition of amyloid deposits in the tissues of animals that have been exposed to a TSE, or are suffering from a neurodegenerative disorder having the characteristics of a spongiform encephalopathy.